T-cell therapy formulation

ABSTRACT

Ex-vivo prepared T-cells are harvested from cell culture conditions and formulated in medium suitable for infusion. The formulation is made by labeling the cells with one or more agents which have reactivity for T-cell surface moieties capable of delivery activation signals upon cross-linking and mixing the labeled cells with biodegradable nanospheres or microspheres coated with a material capable of cross-linking the agents attached to the T-cell surface moieties. Alternatively, the formulation may be made by mixing a population of T-cells with biodegradable nanospheres or microspheres coated with a first material and one or more second materials. The first material binds the second material and the second material has reactivity for surface moieties on the T-cells and the interaction of the second materials with the T-cells causes the activation of the T-cells. In either method, the mixture of T-cells and biodegradable spheres are suspended in a medium suitable for infusion, and the mixture is packaged in a container.

The present application claims priority of U.S. provisional patentapplication Ser. No. 60/549,032, filed Mar. 1, 2004, entitled CellTherapy Formulation Method and Composition.

FIELD OF INVENTION

This invention relates to methods for formulating ex-vivo preparedT-cells for infusion.

BACKGROUND OF THE INVENTION

Cell therapy methods have been developed in order to enhance the hostimmune response to tumors, viruses and bacterial pathogens. Cell therapymethods often involve the ex-vivo activation and expansion of T-cells.Examples of these type of treatments include the use of tumorinfiltrating lymphocyte (TIL) cells (see U.S. Pat. No. 5,126,132 issuedto Rosenberg), cytotoxic T-cells (see U.S. Pat. No. 6,255,073 issued toCai, et al.; and U.S. Pat. No. 5,846,827 issued to Celis, et al.),expanded tumor draining lymph node cells (see U.S. Pat. No. 6,251,385issued to Terman), and various other lymphocyte preparations (see U.S.Pat. No. 6,194,207 issued to Bell, et al.; U.S. Pat. No. 5,443,983issued to Ochoa, et al.; U.S. Pat. No. 6,040,177 issued to Riddell, etal.; U.S. Pat. No. 5,766,920 issued to Babbitt, et al.).

For maximum effectiveness of T-cells in cell therapy protocols, the exvivo activated T-cell population should be in a state of maximalactivation upon infusion. Efforts for developing improved methods forproducing more effective T-cells for use in cell therapy protocols havefocused on the ex-vivo activation methods. However, ex-vivo activatedcells need to be harvested and administered to patients to have atherapeutic effect. The harvesting of the T-cells removes them from theactivating stimuli available in the ex-vivo cultures. Therefore, thelonger the time from harvest to infusion, the lower the quality of theT-cells.

There is a need to develop T-cell formulations for infusion thatmaintain the cells in a state that can maximally orchestrate an immuneresponse to cancer, infectious diseases and other disease states at boththe time of infusion and while circulating in the blood. Efforts tomaintain the activation state of T-cells at the time of infusion havemost commonly involved the formulation of the T-cells with exogenousIL-2. Systemic IL-2 administration to patients has also been used tomaintain the activation state of T-cells post-infusion. However,exogenous IL-2 administration is extremely toxic to patients and has notresulted in significant benefit to patients undergoing T-cell infusions.

SUMMARY OF THE INVENTION

This invention includes ex-vivo prepared T-cells which are harvestedfrom cell culture conditions and formulated in media suitable forinfusion. The method of formulation involves: (1) labeling the cellswith one or more agents which have reactivity for T-cell surfacemoieties capable of delivering activation signals upon cross-linking;(2) mixing the labeled cells with biodegradable nanospheres ormicrospheres coated with a material capable of cross-linking the agentsattached to the T-cell surface moieties; (3) suspending the mixture oflabeled T-cells and coated biodegradable cross-linking spheres in amedium suitable for infusion; (4) packaging the mixture in a suitablecontainer, such as a syringe or an IV infusion bag; and (5) parenteraladministration of said mixture to a patient.

Alternatively, the invention can be accomplished with a formulationmethod which involves: (1) mixing a population of T-cells withbiodegradable nanospheres or microspheres coated with a first materialand one or more second materials, whereas the first material binds thesecond materials and the second materials have reactivity for surfacemoieties on the T-cells, and whereas the interaction of the secondmaterials with the T-cells causes the activation of the T-cells; (2)suspending the mixture of T-cells and the biodegradable spheres in amedium suitable for infusion; and (3) packaging the mixture in asuitable container, such as a syringe or an IV infusion bag; and (4)parenteral administration of the mixture to a patient.

In practice of the present invention, the T-cells intended for infusionare first harvested from the ex-vivo cell culture environment. Theharvested cells are then either immediately formulated for infusion,cryopreserved for later formulation, or shipped to the patient locationfor formulation.

Upon formulation by the method of the present invention, all the T-cellsfor infusion will be maximally activated in a non-toxic deliveryvehicle.

For the purposes of the present invention, all references to T-cellsincludes a population of cells with at least a portion of the cellscontaining T-cells. T-cells are cells which express TCR, including α/βand γ/δ TCRs. T-cells include all cells which express CD3, includingT-cell subsets which also express CD4 and CD8. T-cells include bothnaïve and memory cells and effector cells such as CTL. T-cells alsoinclude regulatory cells such as Th1, Tc1, Th2, Tc2, Th3, Treg, and Trlcells. T-cells also include NKT-cells and similar unique classes of theT-cell lineage.

PREFERRED EMBODIMENTS

The biodegradable spheres of the present invention are preferablymanufactured from aliphatic polyesters, such as poly(lactic acid) (PLA),poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA) orpoly(carprolactone) (PCL), and polyanhydrides. The spheres are producedinto small particle sizes of 0.1 to 500 microns, preferably less than 10microns and most preferably less than 1 micron. Microspheres of thissize range are capable of direct injection into the body by conventionalmethods. It is preferred that the coated spheres be designed to degradein physiological fluids within 7 days, more preferably within 3 days.

The preferred first material for coating on the biodegradable spheres ispolyclonal goat (or sheep) anti-mouse polyclonal antibodies. By way ofexample, this preferred first material can be used to cross-linkmouse-derived monoclonal antibodies, or fragments or geneticallyengineered derivatives thereof, that have specificity for T-cell surfacemoieties. Thus, for example, the mixing of goat anti-mouse coatedmicrospheres (or nanospheres) with human T-cells-labeled with mouseanti-human CD3 and mouse anti-human CD28 mAbs will cause thecross-linking of the mouse mAbs on the human T-cells through the bindingof the goat anti-mouse polyclonal antibody with the mouse mAbs. Thecross-linking of the mAbs causes the activation of the T-cells.Alternatively, the anti-human CD3 and anti-CD28 can also be first bound,preferably in a 50/50 ratio, on the goat (or sheep) polyclonal antibodycoated spheres and mixed with the T-cells. It will be obvious to thoseskilled in the art that many combinations of first materials and secondmaterials can be used to accomplish the objective of cross-linkingsecond agents attached to T-cell surface moieties in order to initiatesignal transduction and activation of T-cells.

The mixture of T-cells with cross-linked surface moieties is suspendedin infusion medium (e.g., isotonic solutions such as normal saline, 5%dextrose, Plasma-Lyte (Baxter) or Normasol (Abbott). In someembodiments, the infusion medium is supplemented with 0.5%-10% humanserum albumen (HSA).

The mixture is preferably adjusted to a final T-cell concentration ofbetween 1×10⁷ to 1×10⁸ cells per ml of infusion media. In a preferredembodiment, 10⁹ T-cells are formulated in 100 ml of infusion media. Theformulation is then packaged in one or more containers, such assyringes, plastic pouches, or plastic bottles.

Although the present invention has been described with reference topreferred embodiments, workers skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the invention.

1. A composition comprising a treatment effective amount of cells, ofwhich a portion are T-cells, whereby said T-cells are contacted with oneor more first agents that ligate a cell surface moiety on at least aportion of the T-cells, and whereby the first agents are cross-linked bya second agent attached to a biodegradable support to activate theT-cells, and whereby said activated T-cells and biodegradable supportsare suspended in a medium suitable for parenteral infusion in anactivated state at a concentration of at least 10⁷ cells per ml of themedium and packaged in a suitable container for administration of saidcomposition to a patient.
 2. The composition of claim 1 whereby thecontainer is collapsible comprising opposing walls of flexible materialand a flexible tube protruding from the container.
 3. The composition ofclaim 1 whereby the container is a syringe.